Development of a new and improved method for safe, site specific, and efficient gene delivery has become a major focus for human gene therapy. To accelerate attaining this goal, we have introduced the concept of hydrodynamic gene delivery. The procedure entails a rapid tail vein injection of a relatively large volume of saline containing plasmid DNA. We demonstrated in rodents that the hydrodynamics-based procedure is superior in gene delivery to hepatocytes. The mechanism underlying the effectiveness of this method involves an elevated hydrodynamic pressure in the liver vasculature, which enhances endothelial permeability and generates transient membrane pores in hepatocytes. Using these earlier findings, we have developed a computer controlled injection device applicable to hydrodynamic gene delivery in human. In the proposed study, we will combine hydrodynamic gene delivery with a well-developed image-guided catheter insertion technique to demonstrate site-specific gene delivery into liver hepatocytes using swine as an animal model. The objective of this study is to establish a minimally invasive, easily repeatable procedure capable of achieving transgene expression at the putative therapeutic level while maintaining acceptable toxicity profiles. In specific aim 1, we will establish the procedure and optimize the injection parameters for gene transfer to individual lobes of the liver. We will confirm that parameters established in rodents are optimal for liver gene transfer in swine. In specific aim 2, we will take a sequential injection approach to transfect the entire liver. The lobe-by-lobe approach aims to develop a procedure for maximal gene transfer to human liver for treatment of liver related genetic diseases. Specific aim 3 is designed to determine the long-term effect of hydrodynamic gene transfer with respect to persistence of transgene expression, effect on tissue toxicity, immune response to transgene product, and tissue distribution of the transgene. In specific aim 4, we will examine the effectiveness of repeated gene transfer. This translational research is designed to validate a newly developed injection device and to establish a procedure applicable to a clinical setting. The objective is to collect data essential for preparation of clinical trials. If successfully accomplished, the proposed study will significantly advance the field of gene therapy and will provide a new technology for gene delivery. Public Health Relevance: In the proposed study, we will combine hydrodynamic gene delivery with a well-developed image-guided catheter insertion technique to demonstrate site-specific gene delivery into liver hepatocytes using swine as an animal model. The proposed study is designed to validate a newly developed injection device and to establish a procedure applicable to a clinical setting. The objective is to collect data essential for preparation of clinical trials. If successfully accomplished, the proposed study will significantly advance the field of gene delivery and provide a new technology for gene therapy.